The basidiomycete fungus Agaricus bisporus (Lange) Imbach var. bisporus produces a mushroom (technically, an agaric basidiome with a pileus, stipe, veil, and lamellae). This Agaricus bisporus “button mushroom” or “portabella mushroom” is a widely and extensively cultivated species of mushroom. Globally, the annual crop has a value of several billion dollars. The mushrooms are grown commercially on specially-prepared compost in enclosed, environmentally controlled spaces. During maturation, the mushroom primordia form and enlarge on a non-nutritive layer called “casing soil” that is applied to cover the compost. The mushrooms' anatomical structures undergo a developmental progression that, if not interrupted, causes (1) the velar layer covering the lamellae (=‘gills’) to stretch and rupture, (2) the lamellae to be exposed to ambient atmosphere, and also to begin to develop deeper and/or darker coloration, (3) the basidial cells lining the lamellae to undergo nuclear fusion (=karyogamy) followed by meiosis, (4) sterigmata (narrow sporogenic structures) and spore-primordia to appear and enlarge on the distal apex of the basidia, (5) one or more haploid postmeiotic nuclei to migrate into each ‘basidiospore’, (6) the spores to develop a dark brown wall pigmentation, and (7) mature spores to be forcibly discharged from the sterigmata and to become airborne. The development and release of fungal spores jointly comprise and define sporulation.
Traditional commercially-used strains of Agaricus bisporus were originally taken directly from naturally occurring mushrooms or from found ‘wild’ composts in which mushroom mycelium was observed. Some of these older strains are still present in culture collections and at least one such line, which produces brown-capped mushrooms, is still in commercial use. Since the 1970's, a number of laboratories have developed techniques for making fusions between two different strains (equivalent terms are stocks, lines, commercial varieties, etc.) to produce novel ‘hybrid’ strains incorporating haploid nuclei from the two different parent strains. Most often, but not universally, the term ‘strain’ is applied to heterokaryotic cultures incorporating two complementary haploid nuclei in a common cytoplasm. A more inclusive general term, culture, includes not only heterokaryotic cultures, but also haploid, homokaryotic cultures, aneuploid cultures, etc.
The Agaricus bisporus mushroom species utilizes two complementary life-cycles that operate concurrently, through basidia and basidiospores (=spores), in each mushroom. In one life-cycle, some spores receive two sexually complementary, postmeiotic haploid nuclei, and these ‘heterokaryotic’ spores can carry out the complete life-cycle from a single germinating spore; however they can only fuse with other spore-cultures poorly. This is an inbreeding system called “pseudohomothallism”, “secondary homothallism”, or “intramixis”, and such spores can be thought of as colonizers. In the other life-cycle, some spores receive only one post-meiotic haploid nucleus, and while such “homokaryotic” spores cannot complete the life-cycle alone, they have the general ability to fuse with and combine genetic material with many other cultures. This is an outbreeding system called “heterothallism” or “heteromixis”, and such spores can be thought of as crossbreeders. After fusion by, for example, two compatible homokaryotic spore-cultures, a heterokaryotic culture incorporating both haploid nuclei and capable of completing the life cycle may result. Less often, there are also produced, in low numbers, spores that may receive two sexually incompatible (or second-division “sister”) post-meiotic haploid nuclei, as taught by Kerrigan et al. Genetics 133:225-236 (1993); these function as homokaryons but are genetically more heterogeneous. Other rare classes of spores include aneuploid spores and spores sufficiently different, cytogenetically, from the ordinary n=1.0 or n+n=2.0 chromosomal complement states, to resist easy classification; deletion and truncation of chromosomes can produce this type of nucleus and spore. However, any viable spore-culture has at least some chance of participation in a fusion event with another culture, which will lead to a novel culture and genotype.
Although spores, some of which germinate to produce the haploid (n) homokaryons desired for hybridization, are most often used as the source of cultures used to construct novel hybrid strains, other sources of homokaryons can be obtained using methods that induce the repartitioning of cellular contents including nuclei; specific techniques include subdividing heterokaryotic culture mycelia (e.g. mechanical reduction methods, including but not limited to, microsurgery or laser surgery to sever hyphal tips or fragment mycelia), regenerating protoplasts from heterokaryotic cultures, etc.
A typical defined fusion arises from the anastomosis plus plasmoogamy of two compatible haploid, homokaryotic cultures, and is achieved in the laboratory by placing (“pairing”) the two cultures in close proximity on a suitable sterile culture medium, and facilitating anastomosis (=hyphal fusion creating a continuous opening through the hyphal cell wall and the plasma membrane) and plasmogamy (=cytoplasmic mixing). Several other combinations of culture types can also result in fusion processes leading to novel hybrid strains, although such methods generally have a lower probability of success and/or result in one or more undefined hybrid heterokaryotic strains. These methods include pairing a heterokaryon culture with a homokaryon culture, pairing two heterokaryon cultures, pairing cultures at least one of which has at least one nucleus which is either aneuploid or is karyotypically ambiguous or indeterminate (i.e., with respect to ploidy), and preparing undefined mixtures of spores, or spores and mycelium together. All of these cases require microbiological methods that are carried out in the laboratory by the experimenter, using axenic manipulation of pure culture materials on sterile culture media and enclosures, to enable anastomosis and plasmogamy to occur.
Any method, including but not limited to those described above, that allows for a reassociation of genetic material from more than one culture and/or spore, and results in a heterokaryon with a novel hybrid (bi-parental) genotype, is capable of producing an equivalent result. The microbiological methods used to obtain, maintain, and transfer cultures including homokaryons, and which enable fusion via anastomosis and plasmogamy between cultures of basidiomycete fungi, will be referred to herein as “hybridization”. By “parent”, a heterokaryotic strain is meant. However, most often culture fusions are attempted between two haploid homokaryons. Homokaryotic cultures are clonally propagated, and can live and grow indefinitely, but biologically they are the functional equivalent of gametes such as sperm or eggs. It is incorrect to also call homokaryons simply “parents”, and so to make the distinction clear, they will be referred to herein as “homokaryon-parents” as set forth in the definitions below.
Naturally occurring “sporeless” mutations are believed to be rare. A small number of mutations negatively affecting sporulation in some basidiomycete fungi are known. This is not unexpected, given that without sporulation, reproduction of the afflicted individual is prevented. Genetic material from such an individual, including any that determines the trait of sporelessness, will not be represented in the next generation of offspring, and thus such genetic determinants of sporelessness, when they arise spontaneously, are likely to decline in frequency in natural populations. In other words, natural selection against sporelessness tends to reduce the frequency of sporeless genetic determinants (i.e., genetic material determining a trait, for example alleles at a genetic locus) in nature. However, if such a hypothetical mutant allele causing sporelessness has a recessive genetic behavior, and is paired with a functional dominant allele in a heterokaryotic (n+n) strain, then sporulation may occur and the ‘masked’ mutant allele can be transmitted to future generations, and may be maintained in the population except when paired (in a heterokaryotic individual) with another recessive allele for sporelessness. It is further taught, as in Zolan et al., Mol. Cell. Biol. 6: 195-200 (1986), that in some cases “those few . . . spores that are produced are never more than about 1% as viable as the more numerous spores from wild-type strains”.
A small number of “sporeless” basidiomycete strains are known in the art to produce basidiomata (e.g. mushrooms) with only a small number of spores. However, none of these known strains belong to the species Agaricus bisporus. For example, Okuda et al., Genome 52(5): 438-446 (2009), teach a “sporeless mutant strain . . . of [Pleurotus] pulmonarius . . . produces an extremely small number of spores”. In the absence of a precise universal definition of “sporelessness” and for purposes of this invention, the trait of “sporelessness,” meaning the “sporeless” condition or phenotype, includes the specific traits of non-sporulation, reduced sporulation, incomplete development of spores, incomplete release of spores, and/or paler lamellae as are further defined below.
An example of a naturally occurring sporeless mutation is known in the basidiomycete fungus Pleurotus ostreatus as set forth in Eger et al., U.S. Pat. No. 4,242,832. That patent teaches a particular process for producing non-recombined (non-post-meiotic) homokaryons (called “monokaryons”, a synonym, in that document) from vegetative heterokaryotic mycelia of basidiomycetes. A sporeless strain designated “42×11” was obtained via inbreeding by fusing two homokaryotic (haploid) single-spore isolates (SSIs) from a single commercial Pleurotus mushroom strain. It is interpreted that a recessive genetic determinant for a sporeless trait was inherited by both of the haploid (n), homokaryotic SSI offspring, and when these two offspring were mated, the resulting inbred heterokaryotic (n+n) strain had a doubly recessive genotype for the postulated gene determining sporelessness, and the sporeless trait was consequently expressed and observed in the phenotype of mushrooms formed by the newly created heterokaryotic strain. A drawback of this method is that such offspring share a single common parent and will be inbred (and thus not “hybrid”, in the sense of having two different parents), and may, for example, be highly likely to be homozygous for deleterious recessive alleles that could negatively affect any important trait of the strain.
More generally, though, such mutations are obtained by mutagenic processes. This was done on Coprinopsis cinereus as taught by Zolan et al., Methods Mol. Biol. 558: 115-27, (2009) and references cited therein. The resulting non-sporulating mutants have been studied by different laboratories, and several different kinds of mutations have been found. In her 1986 article, Zolan et al. wrote that “There are therefore many mutations that could lead to . . . lack of spore formation . . . ” including some that would affect meiosis, and some that would not. One drawback of artificial mutagenesis is that many random mutations are created, rather than a single desired mutation, and the resulting mutagenized strains often have multiple genetic defects, and are unsuitable for purposes other than research.
Mikosh et al., WIPO Publication No. WO01/12850 A1 teach a method of “using a nucleic acid molecule or fragment thereof” for marking alleles of genes in basidiomycete fungi. These ‘Marker Assisted Selection’ methods (=MAS) were and are well-known methods in the art; and the application was abandoned. What Mikosh et al. incidentally demonstrated, using MAS, was one marker that appeared to be linked to one mutation for sporelessness in the fungus Pleurotus ostreatus. Subsequently, also using genetic markers, the Okuda et al. 2009 article proposed a chromosomal map location for an unspecified ‘sporeless’ mutation in the related species Pleurotus pulmonarius. Notably, the method of the present invention does not use or require any nucleic acid molecule or fragment thereof, nor any other marker or MAS technique, to mark alleles of any gene hypothetically associated with the production of a sporeless phenotype in mushrooms of Agaricus. 
Mikosh et al. (2001) further teach, in an entirely imaginary exercise, that their invention “ . . . provides an essentially spore-less mushroom obtainable by a method according to the invention, for example . . . obtained from . . . cultures of basidiomycetes such as . . . Agaricus bisporus . . . ” However, their claimed invention actually required enabling sporeless (either expressed or latent) biological starting material that would necessarily have to have contributed the actual trait for sporelessness. Because no genetic material of Agaricus bisporus capable of determining a sporeless trait in that species was known to Mikosh et al., their invention was not in fact able to provide the sporeless Agaricus bisporus as alleged, and their statement is believed to be a conjecture. It is in fact the invention of the present application that is uniquely and for the first time able to provide a macro-anatomically normal, sporeless Agaricus bisporus mushroom. Further, as noted, the method of our invention does not rely upon MAS methods to accomplish its object.
Mikosh et al. further imagine the possibility of using ‘genetic engineering’ or DNA-mediated transformational methods to silence or ‘knock out’ a gene required for sporulation of basidiomycete fungi. Such future developments are trivially easy for practitioners of the art to imagine, but were never accomplished by Mikosh et al. or, to the Applicants' knowledge, by anyone else. Notably, the method of the present invention does not use genetic engineering methods at all, and explicitly not to silence or ‘knock out’ a gene required for sporulation.
Based on published experiments with Coprinopsis cinereus by Zolan et al. and others, documenting the complexity of the meiotic and sporogenic processes, the number of possible genetic defects that can interrupt the development and release of mature, typical, viable spores, and the way that those often unidentified genes interact with the rest of the organism's genotype, there is an expectation of diversity in both the nature and the degree of “sporeless” phenotypes that may be discovered. Per Mikosh et al. (2001) and Okuda et al. (2009), a few spores might be produced in sporeless strains of Pleurotus. In Coprinopsis, non-viable spores may be produced (Zolan et al. 1986). As explained below, this condition would also be of potential value as it would still be expected to interdict the spread of mushroom viruses by airborne spores, which are believed to be infective only if viable.
The possibility can be anticipated that tiny, aborted spores, or immature unpigmented spores, might be produced. In another instance, mature spores might be developed but not released due to a defect in the spore-release mechanism. The present invention defines ‘incomplete development of spores’ below to include the above conditions and any others that inhibit the production and/or release of mature, typical, viable spores from otherwise typical mushrooms regardless of the quantity of spores.
In the published literature on Agaricus, there are believed to be only a few articles that might appear to have even a remote relationship to the present invention. In fact, these articles describe a fundamentally different phenomenon relating to mutations that produce ‘anatomical monstrosities’ that do not have the familiar anatomy of typical, normal mushrooms. Fritsche, Mush. Sci. 6: 27-47 (1967) described studies on a series of spontaneous mutations in a single Agaricus bisporus culture and a series of its subcultures. In the mutant basidiomata (which were not characteristically (macroanatomically) “agaric” mushrooms in form) the development of entire anatomical structures was prevented. In the first mutation in the sequence, an irregular quasi-mushroom-like structure lacking both lamellae and a stipe (stem) was produced. After a second mutation, these stipeless, nonlamellate bodies became regular in form, somewhat like hens' eggs. Two subsequent mutations led to the production of amorphous masses of undifferentiated tissue not at all resembling mushrooms. Other articles, for example, Umar et al., p. 563-570. In T. J. Elliott [ed.], Science and cultivation of edible fungi, Balkema, Rotterdam (1995), teach similar or other types of irregular ‘monstrosities’ that might hypothetically arise from what may be a same or similar, or a distinct, mutation. In severe cases the monstrosities that are formed lack many anatomical features, among which lamellae may be absent. Technically, basidiomata lacking lamellae and basidia may incidentally be sporeless, but are not macroanatomically normal as defined herein. In contrast, the commercially desired sporeless mushroom is macroanatomically normal, and looks completely familiar to the consumer.
In the absence of meiosis, no genetic recombination will occur in a sporeless strain, and without spores, there is no inherent way for sexual offspring to be produced. Both of these behaviors present obstacles to the creation of sporeless strains, and the lack of available desirable native genetic material causing sporelessness without incurring the deleterious consequences of random mutagenesis to the strain is yet another impediment. Prior to the development of the present invention, no evidence for the existence of the sporeless trait or of natural or artificial genetic determinants for sporelessness in Agaricus were known.
Sporulation of commercially cropped mushrooms is undesirable for several reasons. Spores from mushrooms infected with dsRNA viruses are known to incorporate virus copies and airborne spores can spread the infection within and between facilities, making disease control very difficult. Virus diseases of Agaricus mushroom crops are known to reduce productivity, delay crops, and alter the appearance of product in ways that reduce or eliminate its commercial value. At higher concentrations of mushroom and other fungal spores, such as can occur in enclosed mushroom production facilities, there is a risk of an allergic response and/or respiratory distress in humans. In Agaricus, the spores are a dark ‘chocolate brown’ color (approximately 187A in the RHS color chart system), and dark spores on the surfaces of the lamellae darken the lamellae as seen and contribute to a visual impression of age (overmaturity or lack of freshness) in fresh mushroom product. For specific products with opened mushroom-caps, such as ‘flats’ or ‘portabellas’, the release of spores after harvest and their deposition on other harvested product or packaging can ruin the appearance and commercial value of fresh (‘raw’ or unprocessed) product. Dark spores on lamellae also cause a darkening of moist food dishes incorporating mature mushrooms, often detracting from the appearance of the finished dish.
To counteract many of these problems, mushrooms have traditionally been picked when immature, as ‘buttons’, ‘cups’ or ‘closed caps’. However, the earlier a mushroom is harvested, the less it weighs, and so early harvesting strategies can carry a potential yield penalty and loss of profit. In recent years a market segment for open mushrooms has expanded, in part because mature mushrooms may be perceived to be more flavorful. Commercial production of open mushrooms potentially incorporates all of the negative hygienic and other consequences noted hereinabove.
Mushrooms with lamellae that are paler at any stage of development, relative to and in comparison to typical commercial strains, are highly desirable. Paleness may be obvious or may be established objectively and quantitatively as described herein.
Accordingly, there is a need for new mushroom strain capable of producing visually (macroanatomically) typical mushrooms that have a greatly reduced, or no, capacity for sporulation, by which is meant the production and release of mature, typical, viable spores, as well as acceptable productivity, vigor, timing, and appearance. There is also a need for a method to enable the development of sporeless Agaricus strains, by providing strains with native genetic materials that (1) confer the trait of sporelessness upon strains, that further (2) do not interfere with meiotic recombination, and yet further that (3) via suitable methods can provide recombinant offspring through multiple generations of hybridization. There is yet another need, for a method of obtaining postmeiotic offspring in the absence of sporulation.